{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:22:46Z","timestamp":1785410566376,"version":"3.56.0"},"reference-count":36,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T00:00:00Z","timestamp":1778284800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100011893","name":"INIFAP","doi-asserted-by":"publisher","award":["12443434025"],"award-info":[{"award-number":["12443434025"]}],"id":[{"id":"10.13039\/501100011893","id-type":"DOI","asserted-by":"publisher"}]},{"award":["12443434025"],"award-info":[{"award-number":["12443434025"]}],"id":[{"id":"https:\/\/ror.org\/00r6gdp61","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Postgraduate System of the University Center for Biological and Agricultural Sciences, University of Guadalajara"},{"id":[{"id":"https:\/\/ror.org\/043xj7k26","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Cryopreserved bovine semen represents an accessible source of genetic material due to its widespread use in assisted reproductive technologies and the conservation of genetically valuable animals. However, DNA extraction from spermatozoa within this type of sample remains challenging due to the high protein content and the complex structure of the ejaculate, which can affect DNA yield and quality. The aim of this study was to identify and validate an efficient method for obtaining high-quality DNA from spermatozoa present in cryopreserved bovine semen for molecular diagnostic applications. Five DNA extraction protocols were evaluated: TRIzol\u2122, MagMax\u2122 Nucleic Acid Purification Kit, Rapid DNA\u2122 Fecal\/Soil Microbe Kit, a conventional phenol\u2013chloroform protocol, and a modified phenol\u2013chloroform\u2013isoamyl alcohol protocol. All extracted genetic material was assessed by spectrophotometry (concentration and purity), and DNA integrity was evaluated by agarose gel electrophoresis. Statistical analysis revealed significant differences in DNA concentration among extraction methods (Friedman test, \u03c72 = 22.0, df = 4, p = 0.0002). Post hoc comparisons indicated that the modified phenol\u2013chloroform\u2013isoamyl alcohol protocol yielded significantly higher DNA concentrations compared to selected methods. This protocol showed the highest DNA concentration (1006.2 \u00b1 829.4 ng\/\u03bcL) and favorable purity values, and enabled consistent amplification in both conventional PCR and qPCR assays targeting the \u03b2-actin gene and Tritrichomonas foetus, respectively. These findings suggest that the modified protocol represents a suitable and promising approach for extracting genomic DNA from spermatozoa in cryopreserved bovine semen, with potential applications in molecular diagnostics and reproductive biotechnology.<\/jats:p>","DOI":"10.3390\/mps9030075","type":"journal-article","created":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T12:14:52Z","timestamp":1779452092000},"page":"75","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparative Evaluation of Methods of DNA Extraction from Cryopreserved Bovine Semen for Molecular Diagnostic Applications"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-5752-058X","authenticated-orcid":false,"given":"Carlos A.","family":"Ramos-Jonap\u00e1","sequence":"first","affiliation":[{"name":"Department of MIPPE, Universidad de Guadalajara, CUCBA Campus, Sistema de Postgrado, Camino Ing. Ram\u00f3n Padilla S\u00e1nchez, 2100 Predio Las Agujas, Zapopan 45200, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3474-3289","authenticated-orcid":false,"given":"Lily X.","family":"Zelaya-Molina","sequence":"additional","affiliation":[{"name":"Centro Nacional de Recursos Gen\u00e9ticos-INIFAP, Av. Biodiversidad 2498, Tepatitlan de Morelos 47600, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1663-3457","authenticated-orcid":false,"given":"Luis Felipe","family":"Guzm\u00e1n","sequence":"additional","affiliation":[{"name":"Centro Nacional de Recursos Gen\u00e9ticos-INIFAP, Av. Biodiversidad 2498, Tepatitlan de Morelos 47600, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3095-6869","authenticated-orcid":false,"given":"Edgar I.","family":"Gonz\u00e1lez-Jim\u00e9nez","sequence":"additional","affiliation":[{"name":"Centro Experimental Tecom\u00e1n-INIFAP, Carretera Colima-Manzanillo, km 35, Tecom\u00e1n 28930, Colima, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0820-8052","authenticated-orcid":false,"given":"David","family":"Urb\u00e1n-Duarte","sequence":"additional","affiliation":[{"name":"Centro Nacional de Recursos Gen\u00e9ticos-INIFAP, Av. Biodiversidad 2498, Tepatitlan de Morelos 47600, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7200-8912","authenticated-orcid":false,"given":"Horacio","family":"\u00c1lvarez-Gallardo","sequence":"additional","affiliation":[{"name":"Centro Nacional de Recursos Gen\u00e9ticos-INIFAP, Av. Biodiversidad 2498, Tepatitlan de Morelos 47600, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco J.","family":"Padilla-Ram\u00edrez","sequence":"additional","affiliation":[{"name":"Department of MIPPE, Universidad de Guadalajara, CUCBA Campus, Sistema de Postgrado, Camino Ing. Ram\u00f3n Padilla S\u00e1nchez, 2100 Predio Las Agujas, Zapopan 45200, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Zhang, S., Zhang, Y., Yang, C., Ju, Z., Wang, X., Jiang, Q., Sun, Y., Huang, J., Zhong, J., and Wang, C. (2015). The g.-165 T>C Rather than Methylation Is Associated with Semen Motility in Chinese Holstein Bulls by Regulating the Transcriptional Activity of the HIBADH Gene. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0127670"},{"key":"ref_2","first-page":"79","article-title":"M\u00e9todo r\u00e1pido para la determinaci\u00f3n de fenol y pentaclorofenol en agua potable mediante HPLC con detecci\u00f3n UV","volume":"20","author":"Silva","year":"2013","journal-title":"Rev. Ing. 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